Aberrant signaling by the EGF receptor (EGFR) is responsible for several human cancers, and is the target of a number of therapeutic agents in clinical use and/or trials. Recent studies of the isolated extracellular and kinase domains of EGFR have provided significant insights into the regulation of this important receptor. One of the key lessons, however, has been that the EGFR is allosterically regulated, with contributions from all regions - including the cytoplasmic juxtamembrane (JM) and carboxyterminal (CT) regions that are not well understood and have typically been omitted from experimentally studied constructs. We recently showed that the JM region of EGFR contains an important activation domain, and harbors novel but rare activating mutations found in lung cancer patients. Several studies further indicate that the CT region is required for kinase activation in a manner divorced from its role as the site for receptor autophosphorylation. This region is also thought to contain an auto-inhibitory domain. In this proposal, we will extend our studies of the EGFR JM activation domain (JMAD) to other ErbB receptors in which it is conserved - thus gaining new insight into regulation of ErbB-2 and ErbB- 4. We also propose experiments to elucidate how the large CT region regulates EGFR kinase activity, and whether it does so independently or in concert with the JM region. In a collaborative effort between the Carpenter and Lemmon laboratories, we will combine biochemical analyses of entire receptor intracellular domains (ICDs) with studies of the intact receptors to investigate the physiologic relevance of the JM and CT regions in the control of EGFR (and other ErbB receptors), and their importance for ligand-dependent signaling and mitogenesis. In vitro biophysical and structural analyses will also be pursued in order to gain a detailed mechanistic understanding that will suggest approaches for pharmacological intervention. Finally, the roles of the JM and CT regions will be evaluated in EGF receptor constructs that include known kinase domain- activating mutations that sensitize tumors to clinically employed kinase inhibitors - thus determining how these important regulatory elements cooperate with one another in controlling the intact EGF receptor. Our revised Specific Aims, which we are confident can be completed within two years, are: 1A. To determine whether the JM activation domain is functionally conserved in ErbB-2 and -4. 2. To determine the mechanism of EGFR autoinhibition by the large regulatory CT region. 3. To investigate how JM and NSCLC mutations interact in EGFR activation.